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Showing: 361 - 380 of 895 results

  1. Antibodies 101: Stripping and Reprobing Western Blots

    Type
    Blog Post
    Published
    Aug. 1, 2024, 1:15 p.m.
    ...You’ll want to set up your blot with a polyvinylidene difluoride (PVDF) membrane, which is sturdier and...Western blots are a great tool to identify a protein of interest in a complicated solution like cell ...already run your blot using nitrocellulose, you may decide you have nothing to lose by trying to strip and...abundance of your targets isn’t too different, consider antibody affinity: stronger binding antibodies...matter what stripping methods you throw at it. Ideally, dry your membrane once before any detection steps...(βME) 100–115 mM = 0.7%–0.8% (v/v) Breaks disulfide bonds HCl Adjust pH to 6.7–6.8 Adjust ...addgene.org Western Blot Protocol Western Blot Protocol Video ...

  2. Viral Vectors 101: Chemogenetics

    Type
    Blog Post
    Published
    July 11, 2023, 1:15 p.m.
    ...Chemogenetics has become a popular tool in the field as it provides a noninvasive method for manipulating cell activity... fast-acting optogenetic tools. The advantages provided by chemogenetics are instead that it is noninvasive... crossing the blood-brain barrier.  Another consideration when picking ligands for chemogenetic studies...response you aim to manipulate. It is important to consider that the same receptor can elicit different responses...muscarinic receptor derived DREADDs has been clozapine-N-oxide (CNO). However, concerns have been raised over the...Pharmacokinetic and pharmacodynamic actions of clozapine-N-oxide, clozapine, and compound 21 in DREADD-based chemogenetics... Gomez J, Ramirez M, Hu X, Galvan A, Basu J, Michaelides M, Sternson S (2019) Ultrapotent chemogenetics...

  3. Antibodies 101: Choosing the Right Isotype

    Type
    Blog Post
    Published
    April 6, 2023, 3 p.m.
    ...isotype choice. Target and application should be considered The first question you may have is whether a ...monoclonal primary antibody (A). To boost signal, consider using subclass specific secondary antibodies instead...strength (Fig. 1). Monoclonal primaries If you do decide on a monoclonal antibody you may wonder, “Will ...immunoprecipitations. Your sample species should also be considered when choosing an antibody. In some applications...options are available to you, it may be worth considering an antibody raised in a different species.    ...      Table 1: Isotype considerations when choosing antibodies   Complexity is a key factor...with a distinctly conjugated secondary antibody. Consider too that some secondary antibodies can cross react...

  4. Plasmids 101: In-house NGS Plasmid Quality Control Now at Addgene

    Type
    Blog Post
    Published
    April 6, 2021, 12:30 p.m.
    ...sequence or a sequence provided by the depositor. Depositing labs often provide insert sequences or annotated...to check the quality of other services that we provide, including viral preps and pooled libraries. As...this changeover had no impact on our ability to provide requesting scientists high quality data! Addgene's...culture to prepare it for sequencing, we feel confident that these few minor mismatches usually don’t ...differs from what we would expect given the data provided by the depositing lab, we call these “quality ...Sequencing and Analysis Platform to Assess the Identity of Recombinant Adeno-Associated Viral Preparations...

  5. Reaching out to China: Canton Nucleic Acids Forum (CNAF) 2015

    Type
    Blog Post
    Published
    Dec. 1, 2015, 3:30 p.m.
    ..., loci were identified for 61 such diseases. Now single gene alleles have been identified for 4,163, over...surveillance of repetitive gene families and provide a way to guide inheritance that might be less error prone...plasmids, especially those of the CRISPR variety (see slides from my talk below). Largely organized by Dmitry...of items, only a small number of which I could identify. There were dozens of different mushroom varieties...how the NIH sees the future for genomics, and President Obama’s Precision Medicine Initiative and how ...shRNA silencing of one circular RNA they have identified resulted in a cell migration defect. They have...

  6. Magnetic Control of Proteins: More than a Dream

    Type
    Blog Post
    Published
    Sept. 24, 2024, 1:15 p.m.
    ...magnetoresponsive proteins. Motivated by curiosity Although a wide range of fluorescent proteins and optogenetic tools...possible only in non-biological conditions. So, they decided to search for a magnetic response among other proteins...Hayward et al. 2024 under CC-BY license.   Video 1: Fluorescence movie of E. coli expressing MagLOV...imaged area measures approximately 82 mm across. Video courtesy of Andrew York and Maria Ingaramo.   MagLOV...fluorescent proteins.  Currently, the team is working to identify how MagLOV’s structure changes under magnetic ...microscopy tools for biological applications across a wide range of topics. He embraces open science, self-... for?”, and while Andrew and Maria have lots of ideas and are happy to speculate, they know they can’t...

  7. Simplify Cloning with in vivo Assembly

    Type
    Blog Post
    Published
    Oct. 18, 2018, 12:37 p.m.
    ...with low fidelity polymerases and the cost of synthesizing longer oligos, which could be avoided using restriction...into E. coli for propagation. However, while not widely known, plasmid assembly can be achieved in vivo... of a plasmid, order primers that bind at either side of the undesired portion, designed to amplify the... Mutagenesis is performed by designing primers astride the old codon, amplifying the whole vector. The...homology, so performing more complex procedures can be aided by extending homologous sequences (to 25-30 bp)....colonies for simple cloning procedures. Use high fidelity DNA polymerase This will minimize random errors... blog posts Read our blog post on 5 factors to consider before choosing a cloning method Check out all...

  8. Lentiviral Vector Uses and Overview

    Type
    Blog Post
    Published
    May 19, 2016, 2:30 p.m.
    ...envelope have high infectivity in a wide range of cell types, so they’re ideal for work with primary cells or...envelope protein (Env), usually from VSV-G due to wide infectivity, is often used as a substitute for HIV..., used both for targeted modification and genome-wide screening.   Lentiviral vectors in the clinic Since... check out our lentiviral plasmid and biosafety guides before beginning your experiment. Let us know how...PMCID: PMC4273606. 4. Improved vectors and genome-wide libraries for CRISPR screening. Sanjana NE, Shalem...Genome-Scale, Knockout Screening Read about Genome-Wide Screening Using CRISPR/Cas9 Additional Resources...Resources on Addgene.org Check out Our Lentivirus Guide Learn about Virus Biosafety Find Lentiviral Vectors...

  9. Plasmid Preps: Different Purity, Different Quantities, Different Uses

    Type
    Blog Post
    Published
    June 16, 2020, 1:25 p.m.
    ...plasmid from a bacterial culture, it is important to consider your experiment.  It will dictate the amount of...260 and 230 nm ratios should fall ~2 - 2.2, and provide an indication of purity versus chaotropic agents...agents as guanidine thiocyanate and guanidine hydrochloride used during plasmid extraction. Figure...be entirely endotoxin free. Endotoxin is a lipopolysaccharide of the cell wall of Gram negative bacteria... For these reasons commercially available kits provide extra washing steps to favor the removal of this...want to utilize your plasmid DNA, so that you can decide what will be the optimal type of plasmid preparation...Brush up on molecular biology protocols using our video collection ...

  10. Neuronal labeling with Spaghetti Monster

    Type
    Blog Post
    Published
    Aug. 14, 2018, 12:42 p.m.
    ...recombinant Adeno-associated virus (AAV) has been widely used to deliver genetic tools to the brain because...neuronal circuits. Epitope tags are short antigenic peptide sequences, attached to a protein of interest (POI...simian virus 5-derived epitope (V5), the synthetic peptide FLAG, the synthetic streptavidin-binding strep-...both (Viswanathan, 2015). For Looger’s team, an ideal probe should combine the solubility, cell tolerance...imaging experiments, the second color is usually provided by red fluorescent proteins such as tdTomato or...smFPs can label fine neuronal structures with high fidelity and at lower concentrations than GFP. smFPs enable...N-cadherin in neurons and show that smFPs fusions provided better labels than fusions to 3 or more HA tags...

  11. Antibodies 101: Immunoprecipitation

    Type
    Blog Post
    Published
    Dec. 7, 2021, 2:15 p.m.
    ...When choosing beads, there are several factors to consider. Agarose beads are sponge-like and vary in shape...shape and structure. Their porous surface provides a large area for binding, but antibodies conjugated ...to the capture antibody and is immobilized. The ideal antibody concentration to use will vary between ...antibody is functioning as expected. In order to identify any non-specific interactions, perform parallel... is used to isolate the protein of interest. The wide availability and high specificity of anti-epitope...appealing, but the disadvantages must be carefully considered before using. For example, the tag may affect...routinely use the chromatin IP (ChIP) technique to identify DNA binding proteins involved in histone modification...

  12. Antibodies 101: The Four ELISAs and When to Use Them

    Type
    Blog Post
    Published
    July 25, 2024, 1:15 p.m.
    ...advantages and disadvantages of each, and some considerations when using the method. ELISA basics All ELISAs...methods. For enzymatic signal detection, users provide a substrate that the enzyme converts into a detectable...those studying low abundance antigens may want to consider alternative detection methods.      Figure...conjugated primary antibody. The reporter reacts with a provided substrate and produces a signal that is directly...can be challenging since scientists must first identify two independent antibodies that work well together... of conjugated antibodies   Additional considerations No matter what ELISA method you choose, it ...sample of wild type cells, purified protein, or a peptide. The positive control will confirm that the procedure...

  13. How to Be an Excellent Trainee

    Type
    Blog Post
    Published
    Sept. 26, 2023, 1:15 p.m.
    ...though. Typically, you will identify a ‘scientific mentor’: someone who provides career advice and offers... and identify any gaps or questions you have, then bring them to your mentor. Pro tip! Consider physically... a quality mentor, and rightfully so! They can provide you with high-level career advice, open doors for...Training follow up After your training session (ideally in the next few days or weeks), look over your ...protocol, or maybe you would like to take pictures/videos of a machine set up while your mentor is demonstrating..., your bench mentor’s training time is time set aside just for you – so do your best to show up as your...

  14. Career Insights: Technical Support Specialist

    Type
    Blog Post
    Published
    Nov. 9, 2017, 2 p.m.
    ... live it out, but ideally without asking too many (annoying) questions. Identifying and solving problems...was aware that such roles existed, but did not consider them for myself. I started picturing myself in...Other Addgenies and Their Careers in Our Career Videos What's required for a role in technical support...any incoming requests or problems - there is considerable diversity in the types of questions customers...consuming and/or less technically challenging. Alongside there is a second-line team that takes on cases...problem. It may not always be straightforward to identify those crucial details because there are just so...Visit the Addgene Careers Page Check Out Our Career Videos ...

  15. Tips for CRISPR Gene Editing in Mice

    Type
    Blog Post
    Published
    June 28, 2016, 10:59 a.m.
    ... will outline a simple method for selecting the guide RNA, validating its efficacy in vitro, and using...d like to manipulate in mind. Now it’s time to consider where you would like to attack this gene. Should... When the target sequence was digested by sgRNA guided Cas9 endonuclease, homology dependent repair (HR... It can take roughly 3 months from the day you decide to do a gene modification experiment in mice to...have your F0 generation (barring life’s little accidents and stuff ups). This is a huge improvement on ...production of mouse lines that could be used to study a wide variety of phenomena, it’s possible that we’ll learn...of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases." Genome research 24.1...

  16. Getting the Most from Your Lentiviral Transduction

    Type
    Blog Post
    Published
    April 7, 2016, 2:30 p.m.
    ... in lower lentiviral titers. Another factor to consider is the age of your culture post thaw. In general...lower titer virus. In such cases you may want to consider concentrating your viral prep. Storage can also.... For long-term storage, viral preps should be divided into single-use aliquots, and stored at -80°C. ... start to a successful infection, one point to consider is how the virus is being titered. Most titering... that routinely produce lentivirus may want to consider testing a variety of cation and serum sources .... “Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention...About Viral Vector Elements Learn How to Do Genome-Wide Screens with CRISPR Lentiviral Vectors Additional...

  17. Your Top Requested Plasmid in 2016!

    Type
    Blog Post
    Published
    Jan. 11, 2017, 8:47 p.m.
    ...restriction sites into which you can clone your 20 nucleotide guide sequence of choice (for generation of a gRNA... to the puromycin resistance gene through a 2A peptide. The puromycin resistance gene enables selection...plasmid post transfection. The 2A "self cleaving" peptide causes the ribosome to translate Cas9-2A and the...the Puromycin resistance gene as separate peptides. PX459 This is the vector backbone. This particular...cleavage site). Finally, in addition to the 2A peptide on its C-terminus, SpCas9 is also fused to a 3X...Resources on the Addgene Website Visit our CRISPR Guide Pages Browse Our CRISPR Collection Find gRNA Design...

  18. Why Do I Need an MTA Anyway?

    Type
    Blog Post
    Published
    May 1, 2014, 8:23 p.m.
    ... we set out to streamline this process. We take pride in facilitating the exchange of not just the physical...that allows you to conduct your experiments with confidence. Benefits of the UBMTA: Peace of mind Addgene...University Technology Managers (AUTM). This MTA provides many benefits for both the providing and receiving... lists below for more details. Benefits to the provider (organization / lab that owns the material) ...publish without editorial comment or review by the Provider Limited liability Addgene makes it easy Even...can get tricky. Here’s how Addgene helps: For Providers: One-time Deposit: Simply deposit with Addgene...the UBMTA and are happy to agree to it. Their confidence and familiarity with the UBMTA result in quick...

  19. Easing the Protein Purification Process with pCri

    Type
    Blog Post
    Published
    June 19, 2015, 3:08 p.m.
    ...and stability. TRX and signal peptide tags help proteins with disulfide bonds fold properly. If a TEV ...the subcellular localization and the number of disulfide bonds in your protein - two parameters that affect... and efficient multiplexing. pCri plasmids are divided into two sets, each of which includes plasmids ...designed for these systems may include a signal peptide (SP) that routes the protein through the secretory...secretory pathway to improve folding and disulfide bond formation. If one prefers expression in E. coli, the...thioredoxin A (TRX)-containing vector can improve disulfide bond formation. pCri also includes a special feature...

  20. Supporting Rare Disease Research with BeHeard 2017

    Type
    Blog Post
    Published
    July 20, 2017, 2:30 p.m.
    ... Genomics Institute partners with companies to provide technological resources for those studying rare...be used to accelerate rare disease research and provide hope to patients suffering from these devastating...Schiavi plans on using C. elegans as a model to identify SMN1 interacting partners and perform whole animal...of mesothelioma sufferers. This disease is less widely understood and often misdiagnosed. Like other forms...mesothelioma tumor samples that they have used to identify candidate genes as drivers of peritoneal mesothelioma...Meothelioma Cancer Alliance Website.  We are proud to provide support to Drs Di Schiavi and Nabavi and will continue...., et al. "PP09. 1–2352: Systematic review of incidence and prevalence of spinal muscular atrophy (SMA...
Showing: 361 - 380 of 895 results